PMC

Contact map displaying the number of interacting atom pairs (N_P) between TRPV1 and oxytocin in simulation Pep1 (A) and Pep2 (B), averaged over the simulation length, using a 4Å cut-off. The prominent interactions (defined as N_P >15) are summarized in panel C. The occurrence of hydrogen bonds is displayed in D. In E new cartoon representation of the TRPV1 system, in complex with oxytocin is illustrated; oxytocin and interacting TRPV1 residues are displayed in licorice representation.

A, Oxytocin- and capsaicin-elicited Ca²⁺ responses in DRG neurons obtained from the WT mice (n_exp. = 33). B, Oxytocin- and capsaicin-elicited Ca²⁺ responses in the WT DRG neurons transiently expressing TRPV1 (3 μg) together with GFP (0.5 μg), (n_exp. = 6). C and D panels show oxytocin- and capsaicin-sensitivity of DRG neurons obtained from TRPV1-KO female (n_exp.= 21) and male (n_exp. = 21) mice, respectively. E, Oxytocin- and capsaicin-induced responses obtained from the TRPV1-KO (^−/−) DRG neurons transiently expressing TRPV1 (0.375 μg) along with GFP (0.5 μg), (n_exp. = 6). F, Summary of oxytocin- and capsaicin-induced responses obtained from the WT and WT with transiently expressed TRPV1 channels. G, Pie graphs present distribution of the oxytocin-sensitive and oxytocin-insensitive DRG neurons isolated from the WT-female (n_cells = 284), WT-male (n_cells = 482), TRPV1-KO-female (n_cells = 318), and TRPV1-KO-male mice (n_cells = 318). All error bars stand for ±s.e.m.

Mouse hindpaws were intracutaneously injected under light sevoflurane anesthesia with the vehicle (n = 3), oxytocin (4 μg/paw, n = 3), capsaicin (1.6 μg/paw, n = 6), or a combination of capsaicin and oxytocin (4 μg/paw, n = 6) and nocifensive behavior was quantified as the time spent licking and flinching. Columns represent the amount of time (sec) animals spent licking and flinching the injected paw during the 30 min observation period following the injection (mean ± SEM, p value according to Student’s t-test).

Patch clamp recording show the current obtained from the wild type TRPV1 and mutants. Panel A demonstrates representative whole cell currents recorded by using gap-free protocol with a holding potential at −70 mV. Traces are shown in current densities (pA/pF). Panel B presents the summary of the relative to control current of TRPV1 wt, L635A, D646A, and F649A constructs obtained upon application of 1 μM capsaicin and 10 μM oxytocin, n = 5 for each construct. Panels C and D display heat-induced potentiation of TRPV1 and the mutants upon exposure to 40 °C pulses.

A, Representative single channel current traces of oxytocin-induced (0.5 μM) TRPV1 obtained in the presence of PIP₂ (5 μM), voltages are indicated on the left. B, Representative ramp recording obtained at −150 to +150 mV. C, D, Graphs demonstrate current voltage relationship (C) and voltage dependence (D) of oxytocin-induced TRPV1, n = 12. E, Dose response of oxytocin-TRPV1 activation, EC₅₀ = 0.316 ± 0.02 μM. F, Current voltage relationship of oxytocin- and capsaicin-induced TRPV1 channels. G, Capsaicin induces higher open probability of TRPV1 outward channel activity, as indicated by open probability differences of oxytocin (2 μM) and capsaicin (2 μM) elicited openings at −60 and +60 mV.

A, Current-voltage (I–V) relations obtained from voltage-ramps from −140 to +140mV in presence of vehicle (black), 10 μM Oxytocin (blue) or 1 μM Capsaicin (red). B, (Upper) time course of representative 10 μM Oxytocin and 1 μM Capsaicin responses; (bottom) the overlay of normalized responses showing differences in the desensitization kinetics. C, (left) G–V curves were obtained by plotting peak tail currents at −100 mV in response to voltage-activated steady state currents from −160 to +160 mV in 20 mV increments: Vm values for control 121.66 ± 14.29 (n = 12), for oxytocin 64.22 ± 8.96 (n = 12), and for capsaicin 76.25 ± 21.03 (n = 4); charge z for control 0.85 ± 0.07 (n = 12), for oxytocin 0.75 ± 0.06 (n = 12), and for capsaicin 0.67 ± 0.108 (n = 4); (Right) Representative traces obtained in transfected cells subjected to voltage steps in control conditions (black), or in presence of 10 μM Oxytocin (blue) or 1 μM Capsaicin (red). D, Dose-response curve fitted to concentrations <30 μM. EC₅₀=7.74 ±8.5, h=1.5 ±0.6.

A, Control experiment with10 μM capsaicin-induced TRPV1 activation (n _exp. = 6, n _cells = 81). B, Subsequent applications of 1 and 10 μM of oxytocin, followed by 10 μM capsaicin elicited transient Ca²⁺ responses with oxytocin, but cumulative effect in the presence of both compounds (n _exp. = 9, n _cells = 96). C, Capsazepine significantly inhibited both the oxytocin and oxytocin/capsaicin-induced responses (n _exp. = 6, n _cells = 84, p values in comparison to the control in panel B are the following: oxytocin 1 μM p = 1.6E-7, 10 μM p = 9.3E-7, oxytocin/capsaicin p = 4.25E-7). D, Co-expression of TRPV1-specific siRNA (0.75 μg), and GFP (0.25 μg), significantly inhibited all the responses (n _exp. = 6, n _cells = 66, p values in comparison to the control in panel B are the following: oxytocin 1 μM p = 1.24E-4, 10 μM p = 3.58E-4, oxytocin/capsaicin p = 8.19E-4). E, Control HEK-293 cells non-expressing TRPV1 channels show no responses to any of the agonists (n _exp. = 4, n _cells = 100, p values in comparison to the control in panel B are the following: oxytocin 1 μM p = 1.04E-11, 10 μM p = 1.16E-9, oxytocin/capsaicin p = 1.88E-13). F, The summary presents the mean under all the conditions. All error bars stand for ±s.e.m.
Oxytocin elicits TRPV1 responses in F-11 neuronal cells transiently expressing the channel.​
G, Oxytocin and capsaicin-induced responses on F-11 cells with transiently expressed TRPV1 (0.5 μg) and GFP (0.15 μg) (n _exp. = 6, n _cells = 71). H, Oxytocin and capsaicin-induced responses on F-11 cells with transiently expressed TRPV1 (0.5 μg), GFP (0.15 μg), and OXTRsiRNA (0.4 μg) (n _exp. = 6, n _cells = 60). I, Oxytocin and capsaicin-induced responses on F-11 cells with transiently expressed empty pcDNA vector (0.5 μg) and GFP (0.15 μg) (n _exp. = 6, n _cells = 48, p values in comparison to TRPV1/GFP expressing cells are the following: oxytocin 1 μM p = 0.009, 10 μM p = 2.1E-6, oxytocin/capsaicin p = 2.5E-9). J, The summary presents the mean of Ca²⁺ responses obtained under all the conditions. All error bars stand for ±s.e.m.